Response of Sweet Potato to Substrates and Acclimatization Conditions in the Greenhouse to Produce High-quality Planting Material

Abstract

The sweet potato is a highly important crop in terms of food security worldwide, its nutraceutical properties have increased demand, generating new market opportunities. The availability of disease-free planting material ensures success in establishing this crop, achieving higher yield and better quality. This study aimed to improve the production system of high-quality planting material in greenhouse conditions through the evaluation of three mixtures of substrates based on peat, vermicompost, rice husk, and coconut substrate in different proportions; the best treatment was subsequently evaluated in the acclimatization process of in vitro plants. The substrates were characterized physicochemically and their effect on growth parameters in sweet potato seedlings was determined. For the acclimatization process of in vitro plants, the best mixture of substrates and a humid chamber during the first eight days of growth was compared to the conventional technique. A substrate composed of peat, vermicompost, and rice husk (3:1:1) resulted in the best seedling development. The proposed in vitro plant acclimatization strategy produced seedlings with good growth, high survival rates (92%), and a good multiplication rate (3.53) compared to the conventional strategy (peat without a humid chamber). The use of an optimal substrate and the incorporation of a humid chamber during the first days of growth guaranteed adequate ranges of temperature and relative humidity that kept the vapor pressure deficit of the leaves below critical levels (< 1.2 kPa). Increased efficiency in the production of high-quality planting material with carefully controlled phytosanitary conditions can make an important contribution to improving global disease management strategies in sweet potato cultivation.

Introduction

The sweet potato (Ipomoea batatas (L.) Lam; Convolvulaceae) is recognized as a functional food with nutraceutical properties due to its high content of dietary fiber, carbohydrates, minerals, vitamins, and antioxidant compounds such as phenolic acids, anthocyanins, tocopherols, and β-carotenes (Alam et al., 2020; Donado-Pestana et al., 2012; Grace et al., 2015). These characteristics make sweet potato a healthy crop with increasing consumer demand, creating an opportunity for producers of this crop, who are mostly in developing countries (Mu and Singh, 2019). China is the largest producer of sweet potatoes worldwide, registering a production of more than 48 million tons for the year 2020, followed by Malawi (7 million), Tanzania (4 million), and Nigeria (3 million). In the American continent, the countries with the highest sweet potato production reported in 2020 were the United States (1.5 million), Brazil (0.8 million), Argentina, and Cuba (0.3 million) (FAO, 2022). In Colombia, sweet potato is an emerging crop and to date about 250 genotypes have been reported, of which 34 genotypes have been evaluated in different conditions in the Colombian Caribbean (Burbano-Erazo et al., 2020). Multilocal evaluation and selection processes resulted in the first sweet potato variety registration under national entities, and this genotype corresponds to the Agrosavia-Aurora sweet potato variety. This variety is characterized by its orange root flesh due to the presence of beta carotene (214 ± 21 μg·g⁻¹) and total carotene (246 ± 25 μg·g⁻¹). Under these conditions, it produced a yield of 20 t·ha⁻¹ of fresh tuberous roots (Rosero et al., 2019).

The sweet potato is a crop that multiplies mainly by vegetative propagule propagation (Kim et al., 2015; Qiao et al., 2020; Ssamula et al., 2020). Vegetative reproduction is the main mechanism of transmission of microorganisms and particularly viruses, that cause causing disease propagation, reduced crop yields and significant economic losses (Sastry, 2013). Different types of viruses have been reported to affect sweet potato (Ssamula et al., 2020), viruses transmitted by vegetative propagation techniques are Sweet potato leaf curl virus (Kim et al., 2015), Sweet potato symptomless virus 1 (Qiao et al., 2020) and Sweet potato pakakuy virus (Zhao et al., 2020). The strategy used to prevent the diseases spreading is the production of high-quality planting material that uses seedlings from a selected variety, which after processes of quarantine, cleaning and indexation, are free of pests and diseases and exhibit good growth and development (Sarolia et al., 2018). In sweet potato, high-quality planting material has been used to guarantee good crop development and reduced yield losses caused by accumulated endophyte diseases and pests (Hainzer et al., 2021; Makokha et al., 2020; Mbewe et al., 2021).

Plant tissue culture is a widely used technique for the initial production of quality planting material, since it enables production and multiplication of disease-free seedlings under in vitro conditions, creating the initial material for the scaling process in the production of high-quality planting material. The micropropagation process consists of selecting disease-free mother seedlings. First, the apical meristems are selected and disinfected with certain protocols, which are then placed in a suitable culture medium and controlled growth conditions (Mwanga et al., 2017; Singh, 2018). At the in vitro growth stage, seedlings are exposed to high levels of relative humidity, constant temperatures, low photosynthetic photon flux density, high concentrations of sugar, salts, and growth-regulating substances in the medium, in the absence of microorganisms. In response to these conditions, plants show low rates of transpiration, photosynthesis, absorption of water and nutrients, and a high rate of dark respiration (Chandra et al., 2010; Osório et al., 2013; Shin et al., 2014).

The next step in the production of quality planting material is scaling that is performed in a greenhouse. At this point, in vitro plants are subjected to a hardening process and acclimatization to prepare them to grow under different conditions from those in the laboratory (Deb and Imchen, 2010). Several changes are encountered by in vitro seedlings transferred to an ex vitro acclimatization process; relative humidity and water availability are determining factors to reduce mortality and allow seedling development (Chandra et al., 2010; Gonçalves et al., 2017; Osório et al., 2013; Shin et al., 2014). The main strategy to avoid water loss and maintain adequate relative humidity involves the use of substrates with good moisture retention and the incorporation of humid chambers, respectively (Chandra et al., 2010; Gonçalves et al., 2017). In sweet potato, acclimatization methods have been reported in a mesh house with sandponic substrates (Wanjala et al., 2020), and a mixture of soil, compost and sand (1:1:2) (Beyene et al., 2020) and good results were obtained for seedling development considering the particular conditions in each case. However, determining the best acclimatization conditions for in vitro sweet potato plants is essential to the successful high-quality planting material production process; therefore, the objective of this research was to improve the conventional production system for planting material of sweet potato (I. batatas L.) by recognizing its response to several substrates and acclimatization conditions under greenhouse conditions.

Materials and Methods

Plant material and growth conditions

A new commercial sweet potato variety named ‘Agrosavia-Aurora’, developed by the Corporación Colombiana de Investigación Agropecuaria-Agrosavia for the Colombian Caribbean region was used in this study.

For substrate evaluation, apical cuttings of the orange-fleshed ‘Agrosavia-Aurora’ sweet potato variety were used as plant material. To exclude effects related to initial material conditions on responses to substrates, uniform cuttings with a mean height of 10 cm, total dry weight of 0.5 g, and with a single functional leaf, were selected and used. To eradicate bacterial and fungal pathogens, the plant material was disinfected with a mixture of fungicides-bactericides 58% copper oxychloride (2 g·L⁻¹) and poloxamer iodine (Agrodyne^® SL; Naturezza, Santander, Colombia) (2 mL·L⁻¹). To evaluate acclimatization conditions, in vitro plants at four weeks of age with a mean height of 3.2 cm, leaf area 7.5 cm² and plant dry weight of 0.03 g were used. These plants were grown in a Caron 7300-50 bioclimatic chamber (Caron Products & Services Inc, Marietta, GA, USA). Light intensity was 500 μmol·s⁻¹·m⁻² at a photoperiod of 16/8 h light/dark. The temperature was set at 27°C in the light cycle and 25°C in the dark cycle, and the relative humidity was maintained at 65 ± 10%; the culture medium used as support was medium Murashige Skoog (MS) (Murashige and Skoog, 1962) supplemented with myoinositol (100 mg·L⁻¹) and thiamine (1 mg·L⁻¹) (Abu Zeid et al., 2022). The seedlings were carefully removed from the container and to prevent microbial contamination the roots were disinfected in a 58% copper oxychloride solution (2 g·L⁻¹).

All experiments were performed under greenhouse conditions in a semi-controlled environment where the plant material was isolated to minimize any exposure to pests; however, temperature and humidity conditions were not completely regulated and were influenced by external climatic conditions. Temperatures of 28–34°C and relative humidity of 60–95% were recorded, and these values are related to the climatic conditions of the study area, which corresponds to those observed in the Caribbean zone in Colombia, which is classified as an agroecological zone of Tropical Dry Forest (BsT), according to Holdridge’s life zone classification (Holdridge, 1967). Conditions in the Caribbean area have been reported as suitable for sweet potato crops (Burbano-Erazo et al., 2020; Rosero et al., 2020). The light source was sunlight, the luminosity was 300–600 μmol·m⁻²·s⁻¹, with light/dark photoperiods of 12 hours. Luminosity during the first week of growth was regulated to 20% using 80% commercial shade cloth, and later the seedlings were moved to an area with a luminosity of 65% that was regulated with a 35% commercial shade cloth. Irrigation was supplied manually in the morning, guaranteeing a volume of 30 to 40 mL per plant daily. To prevent microbial contamination a fungicide based on metalaxyl and mancozeb (Ridomil^® Gold MZ 68 WP; Syngenta, Basel, Suiza) (2 g·L⁻¹) was applied 15 days after planting (dap). In addition, plants were supplemented with foliar fertilizer once a week, using a dose of 2 μL·mL⁻¹ per plant of foliar fertilizer that was composed in g·L⁻¹ of total nitrogen (200), phosphorus (100), potassium (50), magnesium (10), total Sulfur (14), manganese (1), copper (2.5), iron (1), boron (1.5), molybdenum (0.03), zinc (5).

Evaluation of substrates for the hardening of sweet potato plants under greenhouse conditions

Different proportions of four substrates types were evaluated; peat as organic material 100% Sphagnum (Pindstrup Plus^®; Pindstrup, Ryomgaard, Denmark) (P), a greenhouse substrate generally used for germination and rooting processes (Ameri et al., 2012), vermicompost (VC), rice husk (RH), and coconut substrate (CS) generating three substrate mixtures (M) as follows: M1 = P:VC:RH (3:1:1 rat); M2 = P:VC (3:2); M3 = P:VC:CS (3:1:1) and as a control a substrate of 100% peat (conventional), was used. The evaluation of substrates was done in plastic germinating trays with dimensions of 39.8 cm, 26.5 cm and 18.1 cm in length, width, and height, respectively, containing 24 alveoli with a capacity of 205 mL for each alveolus. The selection of substrates was made considering their availability in regional markets, so they can be used in sweet potato seed production schemes by farmers. A completely randomized design with three treatments was established. For each treatment, three repetitions were carried out, and for each repetition eight experimental units (plants) were defined. The experiment was repeated under the same conditions one month after planting the first plants.

Destructive sampling after four weeks of planting was used to measure growth parameters such as: height (cm), number of leaves, stem thickness (mm), root length (cm). Leaf area (cm²), fresh and dry weight of stem, leaves, and roots (g). Dry weight was obtained by drying at 60°C for 24–48 h. Leaf area and color composition were estimated by image analysis as previously described (Pérez-Pazos et al., 2021), under the following conditions: RGB images from leaves were taken with a Canon EOS 600D camera configured with similar settings for all treatments. Light, exposition time and the distance between the specimen and the camera were completely controlled. All leaves were dissected and photographed to estimate leaf area. Mature leaves were used to determined color composition by selecting an area of RGB images using color measurement plug-in in Image J software, Fiji version (Schindelin et al., 2012), which used standardized conditions for all measurements to allow reporting of the data in arbitrary units (a.u). Growth rate was calculated by estimating the slope of the height data recorded from 8 (dap) twice a week. The biomass accumulation was determined through the calculation of the leaf biomass fraction (LBF), stem biomass fraction (SBF), and root biomass fraction (RBF). The indices, absolute growth rate (AGR), specific leaf area (SLA), leaf area ratio (LAR), and source-sink relationship (SSR) were calculated according to previous reports (Hunt, 2017; Li et al., 2015; Pérez-Harguindeguy et al., 2016; Pérez-Pazos et al., 2021; Poorter et al., 2012).

Physicochemical characterization of substrates evaluated for the hardening of sweet potato plants

Substrates were chemically evaluated according to the Colombian standard NTC 5167 for phosphorus, potassium, nitrogen, ash, oxidizable organic carbon, moisture content, loss due to volatilization, moisture retention capacity, cation exchange capacity, density, Carbon/Nitrogen, and pH. Physical analysis was performed in triplicate using a stability analysis of aggregates by the Yoder method (Le Bissonnais, 2016). The structure index (SI) was calculated taking into account equation 1, where SI: structure index, W₁:% of stable aggregates with diameter > 2 mm, W_n:% of stable aggregates with diameter < 0.25 mm, W₂, W₃, W₄, … of stable aggregates in each of the size ranges used, between 2 and 0.25 mm in diameter (Cardona et al., 2016). Mean weighted diameter (MWD) was calculated as described in equation 2, where X_i is the mean diameter of the corresponding size fraction and w_i corresponds to percentage by weight of a respective aggregates fraction from a certain size range (Ghezzehei, 2012). Additionally, the contents of fine aggregates (FA) (< 0.5 mm), middle aggregates (MA) (0.5–2 mm) and extreme aggregates (EA) (> 2 mm) were calculated (Ferreira et al., 2019; Tivet et al., 2013).

  

$$\mathit{SI}=\frac{\sum W_2+W_3+W_4\dots }{\sum W_1+W_n}\times 100$$(Equation 1)

  

$$\mathit{MWD}=\sum _{i=1}^n\stackrel{\_}{x_i}\frac{w_i}{100}$$(Equation 2)

Evaluation of acclimatization strategies of in vitro plants in greenhouse conditions

For the acclimatization of in vitro plants, two acclimatization strategies were evaluated. One was the “conventional” strategy that included a substrate normally used for greenhouse hardening processes corresponding to peat. The use of this substrate has been widely reported in processes of acclimatization and the establishment of horticultural crops in greenhouse conditions (Ameri et al., 2012; Jayasinghe et al., 2010). The second acclimatization strategy proposed in this study was named “proposed” where the mixture of substrates that generated the best response in a previous evaluation in sweet potato seedlings was used. This was composed of peat, vermicompost and rice husk (3:1:1), and considering the results obtained in previous investigations for the acclimatization of sweet potato (Beyene et al., 2020), a humidity chamber was used during the first week of growth with to generate better humidity and temperature conditions for the seedlings.

Acclimatization strategies were evaluated in trays of 24 alveoli. A completely randomized design with two treatments was established. For each treatment, three repetitions were included, and for each repetition five experimental units (plants) were defined. Under these conditions, parameters as height (cm), number of leaves, stem thickness (mm), root length (cm), leaf area (cm²), fresh and dry weight of stem, leaves, and roots (g) and relative growth rate (g·day⁻¹) were evaluated after four weeks of cultivation. The survival percentage and the multiplication rate were defined as the proportion of the total number of produced seedlings and the number of initial seedlings. Finally, the multiplication efficiency of super elite material under greenhouse conditions was measured every four weeks based on production of cuttings. In vitro seedlings were established in the proposed acclimatization system, and they were cut at 4, 8 and 12 weeks. Functional nodes were selected, that is, nodes with active leaves, and after cutting, one node was left on the plant for regeneration. The survival percentage (mother plant) and number of nodes produced after each cut were evaluated. Additionally, during the first eight days of growth, humidity and temperature were monitored with an Extech RHT10 USB type Datalogger (Extech Instruments, Nashua, NH, USA). Leaf temperature measurements were performed with a digital thermometer (Extech Industries). The vapor pressure deficit (VPD) was calculated using data on environmental temperature, relative humidity, and leaf temperature, (Monteith and Unsworth, 2013).

Statistical analysis

To compare the physical characteristics of the substrates and to determine the statistical difference between the substrate mixtures and variables of net leaf area, root length and source-sink relationship, a one-way analysis of variance was performed. The method used for differentiation between means was Tukey’s honestly significant difference (HSD) test with 95% reliability. To determine the best substrate to be proposed at the greenhouse level, multivariate analysis based on principal components from growth and development variables was done, using the treatments as a classification criterion to determine the correlation of variables. To identify statistical differences between the in vitro seedling hardening strategies, a comparison was made using the Student’s t-test. R software was used for the statistical analysis (RTeam, 2020). For the analysis of variance, the “onewaytest” package was used (Dag et al., 2018) and for Tukey’s post hoc test, the “TukeyC” package was used. The principal component analysis was performed with the “princomp” function (Venables and Ripley, 2002), and the principal component plot was generated using the “fviz_pca_biplot” function, while the correlation plot was evaluated using the “corrplot” package (Taiyun and Viliam, 2021). The Student’s t-test was performed using the “t.test” function.

Results

Physicochemical characterization of substrates evaluated for the hardening of sweet potato plants

As a result of the chemical analyzes of the substrate mixtures, it was observed that the substrate composed of the M1 mixture composed of P:VC:RH (3:1:1) presented, in comparison with the control (peat), the highest amounts of potassium, nitrogen, phosphorus, ash, moisture retention capacity, cation exchange capacity, density, and pH (Table 1).

Table 1

Chemical analysis of the substrate mixtures evaluated to produce sweet potato seedlings in the greenhouse.

Results of physical substrate analysis related to aggregate stability (Table S1), were used through eq 1 and eq 2 described in the methodology to obtain a structural index and the weighted average diameter index, in addition to the proportion of fine, extreme and average aggregates. Calculated indices showed significant differences among the evaluated substrates except for percentage of fine aggregates (Table 2). The M1 mixture showed significant differences from the rest of the substrates and had the highest structural index. The M1 mixture, presented in comparison with the control, had a high percentage of medium aggregates, low percentage of extreme aggregates, and a low weighted intermediate diameter (Table 2). The mixture M2 (P:VC; 3:2) did not reach statistical difference with the control, having lower values for the structural index and mean aggregates. Highly significant values were obtained for weighted intermediate diameter and extreme aggregates in the M2 substrate compared to M1 and M3 (Table 2).

Table 2

Structural stability characterization indices.

Influence of the substrate on biomass parameters and growth rates in sweet potato during hardening

Foliar development of sweet potato seedlings after four weeks of cultivation under greenhouse conditions was favorable in the M1 (P:VC:RH; 3:1:1) and M3 substrates (P:VC:CS; 3:1:1) (Fig. 1a). Regarding root development under these treatments, radicular structure was more complex with higher root numbers and therefore greater contact area. During the evaluation time, it was not possible to observe the formation of tuberous roots (Fig. 1b).

Fig. 1

Sweet potato seedlings grown under greenhouse conditions in different substrates. a. Photographic record of the seedlings harvested after four weeks of growth. b. Photographic record of root development in the evaluated substrates.

M1 treatment sweet potato seedlings showed better leaf development (P:VC:RH; 3:1:1) compared to M2 (P:VC; 3:2) and the control (peat). Leaf fresh and dry weight, growth rate, leaf mass fraction, and leaf area ratio, were variables significantly influenced by M1 (Table 3). Principal component analysis produced two groups; the variables including leaf number, leaf area, leaf dry weight, leaf mass fraction, leaf area rate, growth rates and absolute growth rate were mainly associated with substrate M1 (P:VC:RH), while the rest of the variables were grouped and associated with substrate M3 (P:VC:CS). Only the stem mass fraction was related to M2 and control treatments (Fig. 2a). The variables associated with M1 produced correlations close to 80%, indicating their synergistic behavior. In contrast, it was observed that the variables associated with M3 had inverse correlations, as in the case of RDW, which is negatively related to SSR and STD (Fig. 2b; Table S2).

Table 3

Response of growth parameters in seedlings grown at the greenhouse level in different types of substrates.

Fig. 2

Principal component analysis between growth variables of sweet potato seedlings grown in different types of substrates. a. Principal components chart. b. Correlations and description of variables.

Image analysis showed significant differences in terms of color composition in red, green and blue pixel values. Significant differences were found in the M1 substrate compared to the control in the blue fraction, which indicates greater absorption at this wavelength. Regarding the red and green fractions, a significant increase was observed in M1 compared to M3, which indicates the presence of a darker green pigmentation (Fig. 3a). Leaf area did not show any statistically significant difference between M1 and M3, but an increase of 17% was observed in M1 (Fig. 3b). The root length variable did not present correlations with other variables; however, it was observed that seedlings grown under the control substrate (100% peat) grew long roots (24 cm) and this was statistically significant compared to the rest of the substrates (Fig. 3c). The use of different mixtures of substrates had an effect on the biomass distribution of sweet potato seedlings, especially on the source-sink relationship (Fig. 3d). Biomass was mainly made up of the source (leaves and stems) when plants were grown under the M2 and M3 treatments. Conversely, biomass under the control treatment was distributed towards the roots, and had a lower value for this index. The M1 mixture generated sweet potato seedlings with an intermediate value for the source-sink relationship index, indicating that the biomass was distributed both in the source and in the landfill, generating a better response in terms of plant development (Fig. 3d).

Fig. 3

Comparison of growth parameters obtained for sweet potato seedlings grown in different types of substrates. a. Mature leaf color. b. Leaf area ratio. c. Root length. d. Source-Sink ratio. a.u: arbitrary units.

Acclimatization strategies of in vitro plants in greenhouse conditions

Specific environmental conditions were produced by the two acclimatization strategies and at 7:00 pm and 7:00 am, the minimum temperature (25 ± 2°C) and maximum relative humidity (86 ± 4%) were recorded. VPD ranged between 0.4 and 0.7 kPa (Fig. 4). From 8:00 am to 05:00 pm, the relative humidity was significantly different between the two evaluated strategies. A significant decrease in relative humidity was found for the conventional strategy until reaching a minimum value of 50 ± 2% in the maximum temperature period (39 ± 3°C (12:00 pm–03:00 pm) and a VPD higher than 1.2 kPa (Fig. 4). In the proposed strategy, the relative humidity showed a decrease, but this was less abrupt, reaching 80 ± 2% in the maximum temperature period (37 ± 3°C) and a VPD lower than 1.2 kPa. The use of a humidity chamber in the proposed strategy allowed efficient conservation of the relative humidity during the period of temperature increase, resulting in a lower VPD.

Fig. 4

Vapor pressure deficit (VPD) values obtained for in vitro seedlings of hardened sweet potatoes under greenhouse conditions. a. Conventional method, 100% peat substrate. Without any cover. b. Proposed method. Substrate peat 60%, vermicompost 20% and rice husk 20%. Humidity chamber for the first eight days.

In vitro plants acclimated under proposed strategy showed a significant increase in growth variables such as height, number of leaves, stem diameter, root length, and particularly, total fresh weight, dry weight, and relative growth rate, reaching increases greater than 100% compared to the conventional method (Table 4). Although no statistically significant differences were observed in variables such as leaf area or fresh and dry weight of leaves, the proposed strategy showed an increase of more than 50% (Table 4). The proposed strategy achieved a higher survival percentage (92%) compared to the control substrate. A significant increase (40%) in the multiplication rate (3.53) of the in vitro plants under the proposed strategy was found in comparison with the conventional strategy (2.5). The greenhouse hardening protocol for these in vitro plants led to greater efficiency both in terms of acclimatization and in the multiplication of high-quality planting material (Fig. 5).

Table 4

Growth parameters of super elite sweet potato seedlings obtained from plants grown under greenhouse conditions with two hardening strategies in vitro.

Fig. 5

Production of quality sweet potato planting material under greenhouse conditions. a. Sweet potato in vitro plants. b. Sowing in germination trays with a mixture of 60% peat substrate, 20% vermicompost, 20% rice husk. c. Humid chamber conditions during the first week of growth. d. Seedling maintenance in greenhouse conditions. e. Super elite sweet potato seedlings obtained after four weeks of growth.

Efficiency evaluation results for the super elite material in multiplication processes under greenhouse conditions indicated that the sweet potato seedlings acclimatized four weeks after planting produced an average of four functional nodes with leaves and a survival percentage of 92% (Fig. 6a). After the first cut (four weeks) the plants with a single functional node remained in the greenhouse for an additional four weeks. Plants eight weeks after planting generated an average of two additional nodes and a survival rate greater than 90%. The plants were subjected to a second cut and remained in the greenhouse for an additional four weeks. Plants 12 weeks after planting only generated one functional node and survival percentages were below 80%, indicating that plant vigor decreased over time, possibly due to the need for nutrient extraction for node production. Therefore, no further cuts were performed (Fig. 6a). A photographic record of the roots indicates that the plants 12 weeks after planting, under greenhouse conditions did not develop tuberous roots (Fig. 6b).

Fig. 6

Evaluation of multiplication efficiency of super elite sweet potato material. a. Survival percentage and number of nodes obtained at 4, 8 and 12 weeks of cultivation determined for super elite sweet potato seedlings during a greenhouse multiplication process. b. Photographic record of aerial and root development of super elite plants in the greenhouse multiplication phase.

Discussion

Influence of the physicochemical substrate composition on biomass parameters and growth rates in sweet potato during hardening

Several differences in the development of orange-fleshed Agrosavia-Aurora sweet potato seedlings under substrate types were found (Fig. 1). The M1 mixture composed of peat, vermicompost, and rice husk in a 3:1:1 proportion achieved the best results for most foliar parameters and growth rate of sweet potato seedlings (Fig. 2). These results can be attributed to the physicochemical properties of the substrate mixture. The chemical characterization of the M1 substrate indicated that it has a high cation exchange capacity compared to the other mixtures, as well as a higher content of potassium, phosphorus, and ash (Table 1). The increase in the cation exchange capacity has been previously reported to improve the mineralization capacity, allowing a greater amount of available nutrients (Tirez et al., 2014). Physicochemical characteristics of the M1 mixture possibly facilitated water and nutrient availability, inducing meristem production and growth in sweet potato seedlings, thus leading to greater biomass and vegetative growth (Li et al., 2015; Poorter et al., 2012). Consistently, a high net leaf area indicated that a significant proportion of accumulated biomass corresponded to photosynthetic tissues, improving the absolute growth rate (Widaryanto and Saitama, 2017). Also, M1 had the highest moisture retention capacity, which indicates better water availability for plants, reducing the risk of drought stress (Karhu et al., 2011; Manns and Martin, 2018).

Physical characterization of evaluated substrates through aggregate stability analysis obtained the structure index; a high value in the structure index indicates a better distribution of the aggregates in the soil with a predominance of macroaggregates. According to the results obtained, the substrate that presented the highest structure index was M1, in which predominantly medium aggregates and mainly macroaggregates were found (0.25–2 mm) (Table 2) (Aksakal et al., 2020). The presence of MA on the M1 substrate contributed to a better distribution of pore size, improved water storage capacity, infiltration rates, surface runoff, hydraulic conductivity, and aeration, favoring root anchorage and nutrient absorption (Aksakal et al., 2020; Ferreira et al., 2019; Ma et al., 2020; Ran et al., 2020). The characteristics of this substrate possibly reduced water loss, avoiding hydric stress and supporting anchoring, acclimatization and development of seedlings (Chandra et al., 2010; Gonçalves et al., 2017).

The M2 mixture (peat and vermicompost in a 3:2 ratio) had a low cation exchange capacity, low moisture retention capacity and according to its physical structure, had the highest values for mean weighted diameter, which indicates the predominance of extreme aggregates (EA) (> 2 mm) (Table 2) (Nimmo and Perkins, 2002). Previous studies showed that EAs are less erodible, giving excellent stability, although the stratification of aggregates in the soil decreases, affecting the substrate pore space (Liu et al., 2019). These characteristics lead to lower water availability and could negatively affect the assimilation of nutrients and therefore plant growth.

Under substrate control with the 100% peat substrate, although the seedlings showed low growth, they developed the longest root length compared with the other mixtures (Fig. 3c), supporting the use of this substrate as one of the most used in germination and rooting processes in the greenhouse (Ameri et al., 2012). However, this study suggested that the incorporation of other substrates such as rice husk that improves the availability of nutrients (Tsai and Chang, 2020), inexpensive coconut substrate (Ameri et al., 2012), and vermicompost in adequate proportions (3:1:1), enhanced the source nutrients and physicochemical characteristics of the substrates to improve growth and biomass accumulation in sweet potato seedlings (Table 3; Fig. 3) (Manh and Wang, 2014).

Acclimatization strategies of in vitro plants in greenhouse conditions

The proposed strategy for in vitro plant acclimatation that included a previously selected substrate (peat, vermicompost and rice husks in a 3:1:1 ratio), and humidity chamber used during the first eight days of growth, guaranteed better plant development and growth (Table 4). The acclimatized seedlings in the proposed strategy presented VPD values in a range of 0.4 and 1.2 kPa (Fig. 4b), values that are within the optimal range (0.4 and 1.37 kPa) defined for vegetables (Shamshiri et al., 2018). This indicates that the substrate conditions and humidity chamber maintained low transpiration levels. This substrate composition, as previously discussed, is related to better humidity retention capacity (Manns and Martin, 2018). In addition, the use of a humidity chamber maintains relative humidity at desired levels that reduce the microenvironmental temperature (Beyene et al., 2020). Under these conditions, evapotranspiration is reduced and VPD is conserved at an adequate level. In contrast, under conventional treatment with a peat substrate and without a humidity chamber, VPD values were higher than 1.37 to 1.8 kPa. Consistently, peat substrate exhibits low humidity retention capacity, and there is a lot of water loss due to the absence of a humidity chamber. These conditions can produce drought stress, and this is evidenced in the high VPD values and low plant survival percentage. An increase in VPD negatively affects the physiological performance of the plants, generating a decrease in stomatal conductance that induces stomatal closure to minimize water loss; therefore, a cascade activated by the increase in perspiration is initiated at a certain VPD threshold. This process affects the photosynthetic rate, causing a reduction in growth (Grossiord et al., 2020). VPD values higher than 1.3 kPa are considered critical for soybean (Glycine max) (Sadok and Sinclair, 2009). In sorghum (Sorghum sp), values higher than 1.6 kPa (Gholipoor et al., 2010) are needed and in sweet potato values higher than 1.9 kPa (Burbano-Erazo et al., 2020). The previous results confirmed that use of a suitable substrate and maintenance of constant temperature and relative humidity are determining strategies in the acclimatization of plants in vitro under greenhouse conditions.

In comparison with other hardening and acclimatization strategies of in vitro sweet potato plants, the proposed strategy, which used a selected substrate mixture and humidity chamber for a week, achieved 92% survival and an average plant height of 12.7 cm (Table 4). These results were significantly better than those reported by Hang et al. (2016), who evaluated a substrate with rice husks and sand without any type of cover, achieving 77% survival of in vitro sweet potato seedlings with a height of 7.5 cm. Similarly, these results were better than those reported by Beyene et al (2020), who achieved a survival percentage of 84% for in vitro sweet potato plants using a hardening strategy with a substrate composed of soil, compost, and sand (1:1:2); the plants were placed in pots covered with transparent plastic bags for one week. The survival results for sweet potato were superior compared to those using in vitro plant acclimatization strategies in others crops. In Tuberaria, the survival percentage in a greenhouse was 84% (Osório et al., 2013), while in Plantago 80% survival was achieved (Gonçalves et al., 2017), both species were acclimatized in a 3:1 (v/v) mixture of peat and vermiculite substrates.

Consistent with previous studies, proposed improvements in substrate mixtures (M1 and M3), which foliar biomass production, and an acclimatization strategy, which reduced seedling mortality, were demonstrated to have a direct influence on the morphological and physiological performance of seedlings, which in vitro plants require to ensure adaptation to new environmental conditions. Production of high-quality planting material requires adequate acclimatization that should consider improvements in substrate development and incorporation of growth chambers and humidity chambers as a treatment prior to direct exposure to greenhouse conditions, thus reducing the impact of drastically changing environmental conditions (Gonçalves et al., 2017; Osório et al., 2013).

The observed morphological differences in seedlings grown under different acclimatization strategies were consistent with those observed in other species. At the morphological level, an increase in leaf length and thickness was observed in Tuberaria seedlings grown under an acclimatization process, as well as leaf pubescence development as a plant strategy against water loss (Osório et al., 2013). Suitable acclimatization conditions ensure high photosynthetic rate values that are associated with a gradual improvement in photosynthetic competence during acclimatization (Osório et al., 2013); this process was supported by the proposed strategy, with high values observed for growth parameters and pigments in leaves estimated by RGB pixel values (Fig. 3a). The levels of photosynthetic pigments under the improved substrate treatment helped plants to respond positively to the change from artificial light to sunlight. This response was also observed in Plantago seedlings, in which the contents of total chlorophyll, chlorophyll a and chlorophyll b increased in the acclimatization stage, improving light absorption capacity and photosynthetic efficiency and increasing the carotenoid content to protect the photosynthetic machinery from photo-oxidative damage (Gonçalves et al., 2017). These results demonstrated that suitable acclimatization conditions increase survival percentages (greater than 80%), improving the capacity of planting material production (Fig. 6). The conditions offered to in vitro plants promoted morphological and physiological changes that supported their adaptation to new conditions and superior plant production. Although sweet potato is recognized for its intrinsic phenotypic plasticity, the proposed strategy achieved an increased survival rate of over 90% in the acclimatization process, offering better conditions for rapid adaptation to ex vitro conditions. The proposed acclimation strategy improved the multiplication rate of sweet potato seedlings by more than 40% compared to the control (Fig. 6), an important aspect in the hardening process of in vitro plants (Deb and Imchen, 2010). These results suggested that improvement in growing conditions using substrates with physicochemical characteristics and suitable acclimatization conditions that favor the establishment of the seedling and reduce the stress of in vitro plants transferring from controlled laboratory conditions to be further exposed to environmental conditions that are generally more severe. The substrate composed of peat, vermicompost, and rice husk (3:1:1) and incorporating a humidity chamber during the first eight days of growth allowed the plants to successfully acclimatize and multiply to obtain high-quality materials in greenhouse conditions.

In conclusion, the mixture composed of peat, vermicompost and rice husk in a ratio of 3:1:1, achieved the best results for the main growth parameters. The response was related to its chemical characteristics such as high cation exchange capacity, higher potassium, phosphorus and ash content and high moisture retention capacity, that facilitate the availability of water and nutrients. Similarly, better physical conditions such as a high structural index, predominance of medium aggregates that contribute to a better distribution of pore size, water storage capacity, infiltration rates and surface runoff, hydraulic conductivity and aeration, favor root anchorage. The improvement in the physicochemical characteristics of the substrates improved meristem production and growth in sweet potato seedlings. This approach can improve mass production systems for high-quality planting materials, along with inclusion of a humidity chamber during the first eight days improved the survival rate to 92%, as well as the multiplication rate (3.53) of the initial in vitro plants.

Acknowledgements

We thanks the research assistants Liliana Llorente and Diris Pernett for their technical support in the production of in vitro plants. We thank Jose Luis Pérez Gamero for his support in the establishing the greenhouse experiments and to Remberto Martinez for his support with the photographic record of sweet potato seedlings.

Literature Cited

•  Abu Zeid,  I. M.,  H. I. A.  Soliman and  E. M. R.  Metwali. 2022. In vitro evaluation of some high yield potato (Solanum tuberosum L.) cultivars under imposition of salinity at the cellular and organ levels. Saudi J. Biol. Sci. 29: 2541–2551.
•  Aksakal,  E. L.,  I.  Angin and  S.  Sari. 2020. A new approach for calculating aggregate stability: Mean weight aggregate stability (MWAS). Catena 194: 104708. DOI: 10.1016/j.catena.2020.104708.
•  Alam,  M.,  S.  Sams,  Z. H.  Rana,  M.  Akhtaruzzaman and  S. N.  Islam. 2020. Minerals, vitamin C, and effect of thermal processing on carotenoids composition in nine varieties orange-fleshed sweet potato (Ipomoea batatas L.). J. Food Compos. Anal. 92: 103582. DOI: 10.1016/J.JFCA.2020.103582.
•  Ameri,  A.,  A.  Tehranifar,  M.  Shoor and  G. H.  Davarynejad. 2012. Effect of substrate and cultivar on growth characteristic of strawberry in soilless culture system. Afr. J. Biotechnol. 11: 11960–11966.
•  Beyene,  B.,  T.  Menamo and  G.  Haile. 2020. Protocol optimization for in vitro propagation of Kulfo, orange flesh sweet potato (Ipomoea batatas) variety using shoot tip culture. African J. Plant Sci. 14: 395–401.
•  Burbano-Erazo,  E.,  C.  Cordero,  I.  Pastrana,  L.  Espitia,  E.  Gomez,  A.  Morales,  J.  Pérez,  L.  López and  A.  Rosero. 2020. Interrelation of ecophysiological and morpho-agronomic parameters in low altitude evaluation of selected ecotypes of sweet potato (Ipomoea batatas [L.] Lam.). Horticulturae 6: 1–22.
•  Cardona,  W. A.,  M. M. B.  Benavides and  W. C.  Montoya. 2016. Effect of chemical and organic fertilizers on the aggregation of a soil cultivated with Musa acuminata AA. Acta Agron. 65: 144–148.
•  Chandra,  S.,  R.  Bandopadhyay,  V.  Kumar and  R.  Chandra. 2010. Acclimatization of tissue cultured plantlets: From laboratory to land. Biotechnol. Lett. 32: 1199–1205.
•  Dag,  O.,  A.  Dolgun and  N. M.  Konar. 2018. Onewaytests: An R package for one-way tests in independent groups designs. R J. 10: 175–199.
•  Deb,  C. R. and  T.  Imchen. 2010. An efficient in vitro hardening technique of tissue culture raised plants. Biotechnology 9: 79–83.
•  Donado-Pestana,  C. M.,  J. M.  Salgado,  A.  de Oliveira Rios,  P. R.  dos Santos and  A.  Jablonski. 2012. Stability of carotenoids, total phenolics and in vitro antioxidant capacity in the thermal processing of orange-fleshed sweet potato (Ipomoea batatas Lam.) cultivars grown in Brazil. Plant Foods Hum. Nutr. 67: 262–270.
• FAO. 2022. Food and Agriculture Organization Corporate Statistical Database (FAOSTAT). Available at https://www.fao.org/faostat/en/#home. Accessed April 5, 2022.
•  Ferreira,  T. R.,  L. F.  Pires,  D.  Wildenschild,  A. M.  Brinatti,  J. A. R.  Borges,  A. C.  Auler and  A. M. H.  dos Reis. 2019. Lime application effects on soil aggregate properties: Use of the mean weight diameter and synchrotron-based X-ray μCT techniques. Geoderma 338: 585–596.
• Ghezzehei, T. A. 2012. Soil structure. In P. Ming Huang, Y. Li, M. E. Sumner (eds.). Handbook of Soil Sciences: Properties and Processes., 2nd ed. vol. 2. Routledge Handbooks. Taylor & Francis Group., UK. 1–17.
•  Gholipoor,  M.,  P. V. V.  Prasad,  R. N.  Mutava and  T. R.  Sinclair. 2010. Genetic variability of transpiration response to vapor pressure deficit among sorghum genotypes. F. Crop. Res. 119: 85–90.
•  Gonçalves,  S.,  N.  Martins and  A.  Romano. 2017. Physiological traits and oxidative stress markers during acclimatization of micropropagated plants from two endangered Plantago species: P. algarbiensis Samp. and P. almogravensis Franco. Vitr. Cell. Dev. Biol.–Plant 53: 249–255.
•  Grace,  M. H.,  A. N.  Truong,  V.  Den Truong,  I.  Raskin and  M. A.  Lila. 2015. Novel value-added uses for sweet potato juice and flour in polyphenol- and protein-enriched functional food ingredients. Food Sci. Nutr. 3: 415–424.
•  Grossiord,  C.,  T. N.  Buckley,  L. A.  Cernusak,  K. A.  Novick,  B.  Poulter,  R. T. W.  Siegwolf,  J. S.  Sperry and  N. G.  McDowell. 2020. Plant responses to rising vapor pressure deficit. New Phytol. 226: 1550–1566.
•  Hainzer,  K.,  C.  O’Mullan,  C.  Bugajim and  P. H.  Brown. 2021. Exploring the design and adoption of a clean seed system for sweet potato in Papua New Guinea. J. Crop Improv. 36: 1–25.
•  Hang,  V. T. T.,  H. T. A.  Thu and  V. D.  Hoa. 2016. Developing an efficient regeneration protocol for sweetpotato, Ipomoea batatas (L.) Lam., using nodal explants. Vietnam J. Agri. Sci. 14: 1491–1501.
• Holdridge, L. R. 1967. Life Zone Ecology. (L. R. Holdridge (ed.)). Tropical Science Center, Costa Rica, 1–206 pp.
• Hunt, R. 2017. Growth Analysis, Individual Plants. In B. Thomas, B. G. Murray, D. J. Murphy (eds.). Encyclopedia of Applied Plant Sciences. vol. 1. Elsevier Inc., Netherlands. 421–429.
•  Jayasinghe,  G. Y.,  I. D. L.  Arachchi and  Y.  Tokashiki. 2010. Evaluation of containerized substrates developed from cattle manure compost and synthetic aggregates for ornamental plant production as a peat alternative. Resour. Conserv. Recycl. 54: 1412–1418.
•  Karhu,  K.,  T.  Mattila,  I.  Bergström and  K.  Regina. 2011. Biochar addition to agricultural soil increased CH4 uptake and water holding capacity—Results from a short-term pilot field study. Agric. Ecosyst. Environ. 140: 309–313.
•  Kim,  J.,  E.-J.  Kil,  S.  Kim,  H.  Seo,  H.-S.  Byun,  J.  Park,  M.-N.  Chung,  H.-R.  Kwak,  M.-K.  Kim,  C.-S.  Kim,  J.-W.  Yang,  K.-Y.  Lee,  H.-S.  Choi and  S.  Lee. 2015. Seed transmission of Sweet potato leaf curl virus in sweet potato (Ipomoea batatas). Plant Pathol. 64: 1284–1291.
•  Le Bissonnais,  Y. 2016. Aggregate stability and assessment of soil crustability and erodibility: I. Theory and methodology. Eur. J. Soil Sci. 67: 11–21.
•  Li,  T.,  E.  Heuvelink and  L. F. M.  Marcelis. 2015. Quantifying the source-sink balance and carbohydrate content in three tomato cultivars. Front. Plant Sci. 6: 416. DOI: 10.3389/fpls.2015.00416.
•  Liu,  M.,  G.  Han and  Q.  Zhang. 2019. Effects of soil aggregate stability on soil organic carbon and nitrogen under land use change in an erodible region in southwest China. Int. J. Environ. Res. Public Health 16. DOI: 10.3390/IJERPH16203809.
•  Ma,  L.,  Q.  Wang and  S.  Shen. 2020. Response of soil aggregate stability and distribution of organic carbon to alpine grassland degradation in Northwest Sichuan. Geoderma Reg. 22: e00309. DOI: 10.1016/J.GEODRS.2020.E00309.
•  Makokha,  P.,  R. T.  Ssali,  B. W.  Wanjala,  S.  Rajendran,  M. A.  McEwan and  J. W.  Low. 2020. Yield potential of sandponically produced sweetpotato (Ipomoea batatas (L.) Lam) pre-basic seed for selected genotypes. Open Agric. 5: 236–242.
•  Manh,  V. H. and  C. H.  Wang. 2014. Vermicompost as an important component in substrate: effects on seedling quality and growth of muskmelon (Cucumis Melo L.). APCBEE Procedia 8: 32–40.
•  Manns,  H. R. and  R. C.  Martin. 2018. Cropping system yield stability in response to plant diversity and soil organic carbon in temperate ecosystems. Agroecol. Sustain. Food Syst. 42: 724–750.
•  Mbewe,  W.,  A.  Mtonga,  M.  Chiipanthenga,  K.  Masamba,  G.  Chitedze,  P.  Pamkomera,  E.  Gondwe,  O.  Mwenye and  F.  Chipungu. 2021. Incidence and distribution of Sweetpotato viruses and their implication on sweetpotato seed system in Malawi. J. Plant Pathol. 103: 961–968.
• Monteith, J. and M. Unsworth. 2013. Principles of Environmental Physics: Plants, Animals, and the Atmosphere—John Monteith, Mike Unsworth—Google Libros, Fourth edi. (J. Monteith, M. Unsworth (eds.)). Elsevier Ltd., Poland, 1–401 pp.
• Mu, T. H. and J. Singh. 2019. Sweet Potato: Chemistry, Processing and Nutrition (T. H. Mu, J. Singh (eds.)). Elsevier. Academic Press, New Zealand, 1–400 pp.
•  Murashige,  T. and  F.  Skoog. 1962. A revised medium for rapid growth and bio assays with tobacco tissue cultures. Physiol. Plant. 15: 473–497.
• Mwanga, R. O. M., M. I. Andrade, E. E. Carey, J. W. Low, G. C. Yencho and W. J. Grüneberg. 2017. Sweetpotato (Ipomoea batatas L.). Genetic Improvement of Tropical Crops, 1st ed. Springer, Cham, Switzerland. 181–218.
• Nimmo, J. R. and K. S. Perkins. 2002. Aggregate stability and size distribution. In J. H. Dane, C. G. Topp (eds.). Methods of Soil Analysis: Part 4–Physical Methods. Soil Science of America Book Series, Madison, Wilconsin, USA. 317–328.
•  Osório,  M. L.,  S.  Gonçalves,  N.  Coelho,  J.  Osório and  A.  Romano. 2013. Morphological, physiological and oxidative stress markers during acclimatization and field transfer of micropropagated Tuberaria major plants. Plant Cell. Tissue Organ Cult. 115: 85–97.
•  Pérez-Harguindeguy,  N.,  S.  Díaz,  E.  Garnier,  S.  Lavorel,  H.  Poorter,  P.  Jaureguiberry,  M. S.  Bret-Harte,  W. K.  Cornwell,  J. M.  Craine,  D. E.  Gurvich,  C.  Urcelay,  E. J.  Veneklaas,  P. B.  Reich,  L.  Poorter,  I. J.  Wright,  P.  Ray,  L.  Enrico,  J. G.  Pausas,  A. C.  de Vos,  N.  Buchmann,  G.  Funes,  F.  Quétier,  J. G.  Hodgson,  K.  Thompson,  H. D.  Morgan,  H.  ter Steege,  M. G.  A van der Heijden,  L.  Sack,  B.  Blonder,  P.  Poschlod,  M. V.  Vaieretti,  G.  Conti,  A. C.  Staver,  S.  Aquino and  J. H. C.  Cornelissen. 2016. New handbook for standardised measurement of plant functional traits worldwide. Aust. J. Bot. 64: 715–716.
•  Pérez-Pazos,  J. V.,  A.  Rosero,  R.  Martínez,  J.  Pérez,  J.  Morelo,  H.  Araujo and  E.  Burbano-Erazo. 2021. Influence of morpho-physiological traits on root yield in sweet potato (Ipomoea batatas Lam.) genotypes and its adaptation in a sub-humid environment. Sci. Hortic. (Amsterdam). 275: 109703. DOI: 10.1016/J.SCIENTA.2020.109703.
•  Poorter,  H.,  K. J.  Niklas,  P. B.  Reich,  J.  Oleksyn,  P.  Poot and  L.  Mommer. 2012. Biomass allocation to leaves, stems and roots: Meta-analyses of interspecific variation and environmental control. New Phytol. 193: 30–50.
•  Qiao,  Q., Z.  Zhang, X.  Zhao, Y.  Wang,  S.  Wang,  Y.  Qin,  D.  Zhang,  Y.  Tian and  F.  Zhao. 2020. Evidence for seed transmission of sweet potato symptomless virus 1 in sweet potato (Ipomoea batatas). J. Plant Pathol. 102: 299–303.
•  Ran,  Y.,  M.  Ma,  Y.  Liu,  K.  Zhu,  X.  Yi,  X.  Wang,  S.  Wu and  P.  Huang. 2020. Physicochemical determinants in stabilizing soil aggregates along a hydrological stress gradient on reservoir riparian habitats: Implications to soil restoration. Ecol. Eng. 143: 105664. DOI: 10.1016/j.ecoleng.2019.105664.
•  Rosero,  A.,  C.  Sierra,  I.  Pastrana,  L.  Granda,  J. L.  Pérez,  R.  Martínez,  J.  Morelo,  L.  Espitia,  H.  Araujo and  C.  De Paula. 2020. Genotypic and environmental factors influence the proximate composition and quality attributes of sweetpotato (Ipomoea batatas L.). Agric. Food Secur. 9: 1–17.
• Rosero, A., I. J. Pastrana Vargas, J. A. García Peña, A. A. Espitia Montes, C. M. Sierra Naranjo, J. A. Sierra Monroy, D. H. Martínez Botello, M. O. Santana Rodríguez, J. L. Pérez Gamero, S. M. Regino Hernández, L. B. Espitia Negrete, H. A. Araújo Vásquez, R. Martínez and J. L. García Herazo. 2019. AGROSAVIA Aurora. Variedad de Batata de Pulpa Anaranjada Para El Caribe Colombiano. Corporación Colombiana de Investigación Agropecuaria (Agrosavia), Monteria, 1–32 pp.
• RTeam. 2020. R: The R Project for Statistical Computing. Available at https://www.r-project.org/. Accessed February 14, 2022.
•  Sadok,  W. and  T. R.  Sinclair. 2009. Genetic variability of transpiration response to vapor pressure deficit among soybean (Glycine max [L.] Merr.) genotypes selected from a recombinant inbred line population. F. Crop. Res. 113: 156–160.
• Sarolia, D. K., D. K. Samadia, B. R. Choudhary and D. Singh. 2018. Production of Quality Seed and Planting Materials. Bikaner, Rajasthan (Indian), 1–38 pp.
• Sastry, K. S. 2013. Seed-Borne Plant Virus Diseases, 1st ed. Springer India, India, 1–327 pp.
•  Schindelin,  J.,  I.  Arganda-Carreras,  E.  Frise,  V.  Kaynig,  M.  Longair,  T.  Pietzsch,  S.  Preibisch,  C.  Rueden,  S.  Saalfeld,  B.  Schmid,  J. Y.  Tinevez,  D. J.  White,  V.  Hartenstein,  K.  Eliceiri,  P.  Tomancak and  A.  Cardona. 2012. Fiji: An open-source platform for biological-image analysis. Nat. Methods 9: 676–682.
•  Shamshiri,  R. R.,  J. W.  Jones,  K. R.  Thorp,  D.  Ahmad,  H. C.  Man and  S.  Taheri. 2018. Review of optimum temperature, humidity, and vapour pressure deficit for microclimate evaluation and control in greenhouse cultivation of tomato: A review. Int. Agrophysics 32: 287–302.
•  Shin,  K. S.,  S. Y.  Park and  K. Y.  Paek. 2014. Physiological and biochemical changes during acclimatization in a Doritaenopsis hybrid cultivated in different microenvironments in vitro. Environ. Exp. Bot. 100: 26–33.
•  Singh,  C. R. 2018. Review on problems and its remedy in plant tissue culture. Asian J. Biol. Sci. 11: 165–172.
•  Ssamula,  A.,  A.  Okiror,  L.  Avrahami-Moyal,  Y.  Tam,  V.  Gaba,  R. W.  Gibson,  A.  Gal-On,  S. B.  Mukasa and  P.  Wasswa. 2020. Factors influencing reversion from virus infection in sweetpotato. Ann. Appl. Biol. 176: 109–121.
• Taiyun, W. and S. Viliam. 2021. R package “corrplot”: Visualization of a Correlation Matrix (Version 0.92). Available at https://github.com/taiyun/corrplot/blob/master/corrplot.Rproj. Accessed February 14, 2022.
•  Tirez,  K.,  C.  Vanhoof,  S.  Hofman,  P.  Deproost,  M.  Swerts and  J.  Salomez. 2014. Estimating the contribution of sampling, sample pretreatment, and analysis in the total uncertainty budget of agricultural soil pH and organic carbon monitoring. Commun. Soil Sci. Plant Anal. 45: 984–1002.
•  Tivet,  F.,  J. C.  de Moraes Sá,  R.  Lal,  C.  Briedis,  P. R.  Borszowskei,  J. B.  dos Santos,  A.  Farias,  G.  Eurich,  D. da C.  Hartman,  M.  Nadolny Jr,  S.  Bouzinac and  L.  Séguy. 2013. Aggregate C depletion by plowing and its restoration by diverse biomass-C inputs under no-till in sub-tropical and tropical regions of Brazil. Soil Tillage Res. 126: 203–218.
• Tsai, C. C. and Y. F. Chang. 2020. Effects of Rice Husk Biochar on Carbon Release and Nutrient Availability in Three Cultivation Age of Greenhouse Soils. Agron. 2020, Vol. 10, Page 990 10: 990. DOI: 10.3390/AGRONOMY10070990.
• Venables, W. N. and B. D. Ripley. 2002. Exploratory Multivariate Analysis. In W. N. Venables, B. D. Ripley (eds.). Modern Applied Statistics with S, 4th ed. Springer, New York, NY, New York. 301–330.
•  Wanjala,  B. W.,  R.  Srinivasulu,  P.  Makokha,  R. T.  Ssali,  M.  McEwan,  J. F.  Kreuze and  J. W.  Low. 2020. Improving rapid multiplication of sweetpotato (Ipomoea batatas L. (Lam) pre-basic seed using sandponics technology in East Africa. Exp. Agric. 56: 347–354.
•  Widaryanto,  E. and  A.  Saitama. 2017. Analysis of plant growth of ten varieties of sweet potato (Ipomoea batatas L.) cultivated in rainy season. Asian J. Plant Sci. 16: 193–199.
•  Zhao,  F.,  L.  Wang,  Z.  Zhang,  Q.  Qiao,  Y.  Qin,  Y.  Wang,  S.  Wang,  Y.  Tian,  D.  Zhang and  X.  Zhao. 2020. Seed transmission of sweet potato pakakuy virus in sweet potato (Ipomoea batatas). J. Gen. Plant Pathol. 86: 205–210.